The Monopolin Complex Crosslinks Kinetochore
Components to Regulate Chromosome-Microtubule
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Corbett, Kevin D., Calvin K. Yip, Ly-Sha Ee, Thomas Walz,
Angelika Amon, and Stephen C. Harrison. “The Monopolin
Complex Crosslinks Kinetochore Components to Regulate
Chromosome-Microtubule Attachments.” Cell 142, no. 4 (August
2010): 556-567.
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http://dx.doi.org/10.1016/j.cell.2010.07.017
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Elsevier B.V.
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Detailed Terms
The Monopolin Complex Crosslinks
Kinetochore Components to Regulate
Chromosome-Microtubule Attachments
Kevin D. Corbett,1 Calvin K. Yip,2 Ly-Sha Ee,3 Thomas Walz,2,4 Angelika Amon,3,4 and Stephen C. Harrison1,4,*
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston,
MA 02115, USA
2Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
4Howard Hughes Medical Institute
*Correspondence: harrison@crystal.harvard.edu
DOI 10.1016/j.cell.2010.07.017
SUMMARY
The monopolin complex regulates different types
of kinetochore-microtubule attachments in fungi,
ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic
attachment in Schizosaccharomyces pombe mitosis.
In addition, the monopolin complex maintains the
integrity and silencing of ribosomal DNA (rDNA)
repeats in the nucleolus. We show here that the
S. cerevisiae Csm1/Lrs4 monopolin subcomplex
has a distinctive V-shaped structure, with two pairs
of protein-protein interaction domains positioned
10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore
proteins: Dsn1, a subunit of the outer-kinetochore
MIND/Mis12 complex, and Mif2/CENP-C. Csm1
point-mutations that disrupt kinetochore-subunit
binding also disrupt sister chromatid co-orientation
in S. cerevisiae meiosis I. We further show that the
same Csm1 point-mutations affect rDNA silencing,
probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4
functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid
co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis,
and crosslinking rDNA repeats to aid rDNA silencing.
INTRODUCTION
Mitosis and meiosis are related processes in which duplicated
eukaryotic chromosomes segregate to daughter cells or
gametes (Lee and Amon, 2001; Marston and Amon, 2004;
Nasmyth, 2001). In mitosis, chromosomes replicate and the
resulting sister-chromatid pairs are held together along their
length by cohesin complexes. Associated with the centromere
556 Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc.
of each chromatid is a kinetochore, a specialized protein
assembly that captures microtubules (MTs) of the mitotic
spindle. In early mitosis, each sister chromatid pair becomes
‘‘bi-oriented’’ when its kinetochores capture MTs extending
from opposite spindle poles. Once all chromatid pairs are properly attached, cleavage of the cohesin links between sisters
allows chromosome segregation and subsequent cell division.
DNA replication and cell division strictly alternate in mitosis,
but in meiosis, DNA replication is followed by two successive
divisions to yield four haploid gametes. During meiotic prophase,
homologous chromosomes align and form crossovers that hold
them together. This organization allows for bi-orientation and
segregation of homologs in meiosis I, followed by segregation
of sister chromatids in meiosis II (Lee and Amon, 2001; Marston
and Amon, 2004; Nasmyth, 2001). Thus, although sister chromatids bi-orient and segregate from each other in mitosis and
meiosis II, they instead co-orient and segregate together in
meiosis I.
In the budding yeast Saccharomyces cerevisiae, sister chromatid co-orientation in meiosis I depends on the four-protein
monopolin complex (Mam1, Csm1, Lrs4, and Hrr25/casein
kinase 1), which localizes to centromeres from meiotic prophase
through metaphase I (Monje-Casas et al., 2007; Petronczki et al.,
2006; Rabitsch et al., 2003; Toth et al., 2000). Mam1 is expressed
specifically in meiosis (Toth et al., 2000) and associates at
centromeres with the ubiquitous kinase Hrr25 (Petronczki
et al., 2006). The remaining subunits, Csm1 and Lrs4, form a
complex that resides in the nucleolus during interphase and
relocalizes to centromeres during meiotic prophase, accompanied by phosphorylation of Lrs4 (Huang et al., 2006; Katis
et al., 2004; Lo et al., 2008; Matos et al., 2008; Rabitsch et al.,
2003). Robust centromeric localization of Csm1/Lrs4 requires
Mam1 (Rabitsch et al., 2003). It has been proposed that the
monopolin complex crosslinks and/or co-orients sister kinetochores in meiosis I, so that they attach to MTs extending from
the same spindle pole (Monje-Casas et al., 2007). Although
monopolin complex subunits have not been identified outside
of fungi, the concept of sister kinetochore ‘‘fusion’’ in meiosis I
may have parallels in higher eukaryotes: in maize meiosis I,
for example, inner kinetochores of sister chromatids can be
Table 1. Molecular Mass Determinations from Sedimentation
Equilibrium Centrifugation
Protein/Complex
Csm1 full-length
Observed
MW (kDa)
43.2 ± 0.9
Calculated
MW (kDa)a
43.5
Oligomeric
State
2 Csm1
Csm1 1–181
43.4 ± 1.2
41.2
2 Csm1
Csm1 69–190
29.1 ± 2.3
28.2
2 Csm1
Pcs1 full-length
53.4 ± 1.7
51.8
2 Pcs1
Pcs1 85–222
30.1 ± 2.3
32.2
2 Pcs1
Csm1/Lrs4 full-length
172.3 ± 4.7 165.6
4 Csm1 + 2 Lrs4
Csm1/Lrs4 1–130
120.4 ± 3.1 117.6
4 Csm1 + 2 Lrs4
Csm1/Lrs4 1–102
108.5 ± 3.0 111.0
Csm1 1-181/Lrs4 2–30
91.3 ± 3.1
89.7
4 Csm1 + 2 Lrs4
4 Csm1 + 2 Lrs4
Pcs1/Mde4 full-length
164.2 ± 7.8 198.9
4 Pcs1 + 2 Mde4
Pcs1/Mde4 1–231
160.2 ± 3.6 156.9
4 Pcs1 + 2 Mde4
Pcs1/Mde4 1–125
127.0 ± 2.7 132.5
4 Pcs1 + 2 Mde4
Pcs1/Mde4 1–77
108.2 ± 4.1 121.0
4 Pcs1 + 2 Mde4
a
Calculated MW is the expected molecular mass of a complex with the
stoichiometry listed in ‘‘Oligomeric State.’’
resolved by fluorescence microscopy, whereas their outer kinetochores appear fused (Li and Dawe, 2009).
Orthologs of the monopolin subunits Csm1 and Lrs4 are
present in the fission yeast Schizosaccharomyces pombe
(Pcs1 and Mde4, respectively) and also cycle between the
nucleolus and kinetochores (Gregan et al., 2007; Rabitsch
et al., 2003). These proteins inhibit merotelic attachment
(capture of a single kinetochore by MTs from opposite spindle
poles) during mitosis, but they do not contribute to sister
chromatid co-orientation in meiosis I. (An unrelated protein,
Moa1, is important for ensuring meiosis I sister co-orientation
in S. pombe, probably by modifying cohesin-complex function
near centromeres [Yokobayashi and Watanabe, 2005]). Although
S. cerevisiae kinetochores capture a single MT, S. pombe and
higher eukaryotes assemble larger kinetochores that capture
multiple MTs (2–4 in S. pombe [Ding et al., 1993] and 15–30 in
metazoans [McEwen et al., 1997]). In this context, Pcs1 and
Mde4 have been proposed to organize S. pombe kinetochores
by clamping together adjacent MT-binding sites (Gregan et al.,
2007). In addition, Pcs1 and Mde4 have recently been shown
to localize to the mitotic spindle in anaphase, revealing another
potential function for monopolin in anaphase spindle elongation
and stability (Choi et al., 2009).
During interphase, S. cerevisiae Csm1/Lrs4 and S. pombe
Pcs1/Mde4 are both in the nucleolus, where they have been
shown in S. cerevisiae to be important for maintaining the ribosomal DNA (rDNA) (Gregan et al., 2007; Huang et al., 2006;
Mekhail et al., 2008). The repetitive rDNA array (100–200 copies
of a 9.1-kb repeat in S. cerevisiae) is normally kept in a silenced,
heterochromatin-like state by a network of rDNA-associated
proteins, including Fob1, Tof2, Csm1/Lrs4, and the RENT
complex (Regulator of nucleolar silencing and telophase exit),
which contains Net1/Cfi1, Cdc14, and the Sir2 histone deacetylase (Huang et al., 2006; Huang and Moazed, 2003). The rDNA is
also protected from unequal sister chromatid exchange (USCE),
which can lead to addition or deletion of repeats within the rDNA
(Sinclair and Guarente, 1997). USCE is suppressed by Csm1/
Lrs4 (Huang et al., 2006), the inner-nuclear membrane proteins
Heh1 and Nur1 (Mekhail et al., 2008), and the condensin complex
(Johzuka and Horiuchi, 2009), in addition to Sir2 (Huang et al.,
2006; Smith and Boeke, 1997; Smith et al., 1999). With the
exception of Sir2, which independently contributes to USCE
suppression, these proteins appear to tether rDNA repeats to
the nuclear periphery, sequestering them from recombination
factors (Mekhail et al., 2008), and they may also clamp sister
chromatids together in register (Brito et al., 2010; Johzuka and
Horiuchi, 2009). Thus, although Csm1/Lrs4 contributes to both
rDNA silencing and USCE suppression, it acts with distinct
sets of proteins in these different processes, raising the question
of whether a common mechanism underlies these activities.
The regulatory functions of monopolin described above
suggest that Csm1/Lrs4 and the orthologous Pcs1/Mde4 are
molecular crosslinkers, joining MT-binding elements at kinetochores and rDNA repeats in the nucleolus. We report here that
S. cerevisiae Csm1 and Lrs4 form a complex with a distinctive
‘‘V’’ shape, which positions two pairs of protein-protein interaction domains 10 nm apart. We find that a conserved surface
patch on these domains binds two kinetochore subunits: Dsn1,
a subunit of the outer-kinetochore MIND/Mis12 complex, and
Mif2/CENP-C. Point-mutations in this conserved surface disrupt
both Dsn1 and Mif2 binding in vitro and cause bi-orientation of
sister chromatids in meiosis I. These data are consistent with
a model of monopolin as a crosslinker that clamps kinetochores
together to enforce co-orientation in S. cerevisiae meiosis I and
inhibit merotelic attachment in S. pombe mitosis. We also find
that Csm1 interacts with the nucleolar protein Tof2 through the
same conserved surface that interacts with Dsn1 and Mif2,
and that mutating the Csm1 surface patch also disrupts rDNA
silencing. These mutations do not, however, affect the rate of
unequal sister chromatid exchange, demonstrating that Csm1/
Lrs4 has two biochemically separate roles in the maintenance
of rDNA. Overall, our data show that Csm1/Lrs4 is a molecular
crosslinker that regulates kinetochore-microtubule attachment
and helps preserve rDNA integrity.
RESULTS
Structure of Csm1
We purified full-length S. cerevisiae Csm1 and S. pombe
Pcs1 proteins, as well as truncations lacking the bulk of their
N-terminal regions, which are predicted to form coiled-coils
(Gregan et al., 2007; Rabitsch et al., 2003). By sedimentation
equilibrium analytical ultracentrifugation, we found that both
constructs of Csm1 and Pcs1 are homodimers in solution
(Table 1). We obtained crystals of both full-length S. cerevisiae
Csm1 and its isolated C-terminal domain (residues 69–181 of
190). We determined the structure of the C-terminal domain
to 2.35 Å resolution using anomalous diffraction methods with
selenomethionine-derivatized protein (see Table S1, available
online, for crystallographic statistics), and we then determined
the structure of the full-length protein to 3.4 Å resolution by
molecular replacement. The structures show that Csm1 has
a 12-nm long, N-terminal coiled-coil (residues 3–82), and a
Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc. 557
Figure 1. Structure of Csm1
(A) The Csm1 dimer. One chain is shown in orange (N-terminal coiled-coil, residues 3–82) and dark blue (C-terminal globular domain, residues 83–181), and the
other is shown in gray.
(B) Secondary structure diagrams for Spc24, Spc25, and Csm1, illustrating their common fold (gray) and embellishments in Spc25 (pink) and Csm1 (dark blue).
Secondary-structure elements are labeled according to their position in Csm1.
(C) Structural overlay of the globular domains of Csm1 (dark blue) and Spc25 (gray/pink, colored as in (B); PDB ID 2FTX; Wei et al., 2006). The root-mean-squared
distance calculated for 57 Ca positions (out of 90) is 1.72 Å.
(D) Upper panel: bottom view of the Csm1 globular domain dimer, colored according to amino acid conservation among all identifiable Csm1/Pcs1 orthologs in
fungi (purple = well conserved, light blue = highly variable; for sequence alignment showing conservation, see Figure S1). Lower panel: zoom-in onto the
conserved surface boxed in (A), showing the underlying amino acid residues.
See also Figure S1 and Table S1.
C-terminal globular domain (residues 83–181) containing three
a helices and five b strands (Figure 1A). The b sheet of each
monomer wraps around the N-terminal a helix of its dimer
mate, forming an intimate dimer interface.
Csm1 is a structural relative of the kinetochore proteins
Spc24 and Spc25 (Figures 1B and 1C) (Wei et al., 2006). These
proteins are paralogs that form a heterodimer similar to the
Csm1 homodimer and constitute the inner half of the conserved
Ndc80 kinetochore complex (Joglekar et al., 2006; Wei et al.,
2005). The similarity of the tertiary and quaternary structures of
Csm1 and Spc24/Spc25 imply a common evolutionary origin,
despite their very low sequence identity (<15%). The C-terminal
globular domains of Spc24 and Spc25 are thought to connect
the Ndc80 complex with proteins of the inner kinetochore,
suggesting that this domain of Csm1 may also be a protein interaction module. Inspection of amino acid conservation among
41 fungal Csm1/Pcs1 orthologs reveals a conserved surface patch
on the face directly opposite the coiled-coil, bordering a-3, b-5,
and a-4 (Figure 1D; Figure S1). The patch faces away from the
558 Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc.
dimer interface, so that the Csm1 dimer has two conserved
surfaces centered 3 nm from each other. Because both a-3
and b-5 are located in a Csm1-specific insertion in the globular
domain, this surface is not present in Spc24/Spc25. Therefore,
it is unlikely that Csm1 and Spc24/Spc25 have a common binding site at kinetochores, despite their overall structural similarity.
Structure of the Csm1/Lrs4 Complex
S. cerevisiae Csm1 and Lrs4 are known to interact through their
N-terminal coiled-coil regions (Rabitsch et al., 2003). Coexpression of Csm1 with full-length Lrs4 (347 residues) or with an isolated N-terminal segment (residues 1–130 or 1–102) yields
a complex with four copies of Csm1 and two copies of Lrs4
(Table 1). Coexpression of S. pombe Pcs1 and Mde4 also yields
a complex with 4:2 stoichiometry, with the N-terminal region of
Mde4 (residues 1–77 of 421 is the smallest segment we have
tested) sufficient for complex formation (Table 1; Figure S2).
We obtained crystals of the complex between S. cerevisiae
Csm1 and residues 1–102 of Lrs4, which diffracted anisotropically
Figure 2. Structure of the Csm1/Lrs4 Complex
(A) Diagram of Csm1 and Lrs4 polypeptide chains. Domains of Csm1 are colored as in Figure 1; residues 1–33 of Lrs4 are in green. For Lrs4, predicted coil-coil
(residues 54–82) is in gray, and the motif conserved between Lrs4 and S. pombe Mde4 (Gregan et al., 2007) in red; a blue arrowhead indicates a lysine/argininerich motif (K/R). The gray arrow indicates the interacting regions of the proteins.
(B) Orthogonal views of the (Csm1)4:(Lrs4)2 complex, colored as in (A). Residue numbers of the two Lrs4 a helices are marked. Although Lrs4 residues
34–102 were present in the complex as crystallized, they were disordered in the electron density maps. In the crystals, the two Lrs4 a helices extend into a solvent
channel large enough to accommodate these disordered regions (not shown).
(C) Electron density surrounding the Lrs4 a helices. Refined 2Fo-Fc density (1.2 s) is in gray, and anomalous difference density from an Lrs4 Leu8/Met
selenomethionine (Se-Met) dataset (4.0 s), is in red. The Ca-atom of Leu8 is shown as a sphere. There is a single strong anomalous difference-density peak
directly between the helices, which probably represents the anomalously scattering Se atoms of both Se-Met residues in the Lrs4 dimer.
(D) Electron micrograph of negatively stained Csm1/Lrs4 1–102 complex, with representative particles circled.
(E) Electron micrograph of the full-length nucleolar Csm1/Lrs4 complex, with representative particles circled.
(F) Representative class averages of the full-length Csm1/Lrs4 complex are shown side-by-side with matched resolution-filtered projections of the Csm1/Lrs4
1–102 crystal structure. For more information on the assembly and purification of Csm1/Lrs4 and S. pombe Pcs1/Mde4 complexes, see Figure S2.
See also Figure S2 and Table S1.
to between 6.0 Å (along the a* and b* reciprocal unit-cell axes) and
3.9 Å (along c*) resolution, and we determined the structure by
molecular replacement. We located two Csm1 homodimers per
asymmetric unit and found that the N-terminal coiled-coils of
these two dimers sandwich two closely packed, parallel, 30
amino acid a helices, creating a V-shaped complex that positions
the two pairs of Csm1 globular domains 10 nm apart (Figures 2A
and 2B).
Our electron density maps indicated that two copies of a 30
amino acid region of Lrs4 might be sufficient for complex forma-
tion with Csm1, but the low resolution precluded direct assignment of sequence to this electron density. From sequence
conservation and secondary structure predictions, it appeared
that the density probably represented two N-terminal segments
of Lrs4, with the remainder of the protein (approximately residues 34–102) disordered in our crystals. Mixing Csm1 with
a peptide containing Lrs4 residues 2–30 results in a stable
complex with the expected molecular mass for a 4:2 complex
(Table 1; Figure S2). This complex also forms crystals isomorphous to those of the complex with Lrs4 1–102 (data not shown),
Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc. 559
supporting the inference that Lrs4 residues 34–102 are disordered in the latter crystals and hence do not contribute to the
diffraction intensities. We established the orientation and
sequence register of the Lrs4 a helices using selenomethionine
anomalous scattering from a construct containing a L8/M
mutation in Lrs4. We observed a single anomalous difference
peak in the electron density maps, directly between the two
Lrs4 a helices (Figure 2C); at this resolution (6.0/3.9 Å), the single
peak probably represents the anomalous scattering of both Se
atoms in the Lrs4 dimer. This assignment indicates that the
disordered C-terminal region of Lrs4 extends outward from the
base of the observed V.
We also examined the architecture of the native Csm1/Lrs4
complex using negative-stain electron microscopy. We purified
Csm1/Lrs4 from mitotically cycling S. cerevisiae by TAP-tagging
Lrs4, thus obtaining a near-native nucleolar form of Csm1/Lrs4.
Individual particles of this complex, as well as of the recombinant
Csm1/Lrs4 1–102 complex, show a clear V shape (Figures 2D
and 2E), and class averages of the native full-length complex
can be matched with 2D projections of the X-ray structure
(Figure 2F). We can draw two conclusions from the close correspondence of the projections and class averages. First, the
conformation of the complex is stiff and not grossly affected
by the packing in our crystals. Second, the bulk of the two
Lrs4 subunits is either largely disordered in solution or flexibly
linked to the rest of the complex. This conclusion is consistent
with our observation that residues 34–102 are disordered in
the crystal structure of Csm1/Lrs4 1–102. The C-terminal region
of Lrs4 is nonetheless crucial for regulation of monopolin: phosphorylation and dephosphorylation of Lrs4 (and its S. pombe ortholog Mde4) is needed for localization and activity at kinetochores (Choi et al., 2009; Katis et al., 2004; Lo et al., 2008;
Matos et al., 2008), and even a small C-terminal deletion of
Lrs4 (23 residues) compromises function in the nucleolus
(Johzuka and Horiuchi, 2009).
Csm1 Binds Two Kinetochore Subunits
Previous attempts to identify direct binding partners of Csm1/
Lrs4 using TAP-tagging and mass spectrometry have not identified any kinetochore subunits, which are present in relatively low
abundance in the cell (Huang et al., 2006; Petronczki et al., 2006).
The kinetochore proteins Ctf19 and Dsn1 were recently identified
as potential Csm1 binding partners by a large-scale two-hybrid
screen focused on S. cerevisiae kinetochore proteins (Wong
et al., 2007). Ctf19 is part of the COMA complex, which is not
well-conserved in higher eukaryotes, whereas Dsn1 is a component of the highly conserved MIND/Mis12 complex (Cheeseman
and Desai, 2008). In addition, a recent study identified an interaction between S. pombe Pcs1 and the inner-kinetochore protein
Cnp3/CENP-C (S. cerevisiae Mif2), also by yeast two-hybrid
analysis (Tanaka et al., 2009). We tested binding of in vitrotranslated and [35]S-labeled Dsn1, Ctf19, and Mif2 to several
constructs of Csm1, using a Ni2+-affinity pull-down assay.
Although Ctf19 did not bind Csm1 (data not shown), both Dsn1
and Mif2 bound to full-length Csm1 and to the isolated C-terminal
globular domain, but not to the isolated N-terminal coiled-coil
region (Figure 3A). Because Csm1 has previously been shown
to interact with the monopolin subunit Mam1 (Rabitsch et al.,
560 Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc.
2003), we also tested binding of this protein to our Csm1
constructs and found that Mam1 binds specifically to the
C-terminal globular domain of Csm1 (Figure 3A). We next tested
whether Dsn1, Mif2, or Mam1 binding was affected by point
mutations in the conserved hydrophobic surface patch on the
Csm1 globular domain. None of the mutations tested affected
Mam1 binding (Figure 3A), but three of them, Y156/E, L161/D,
and L161/K, substantially reduced binding of both Dsn1 and
Mif2 (Figure 3A). These results indicate that both Dsn1 and Mif2
contact the conserved surface patch on Csm1 and may even
compete for a common binding surface, whereas Mam1 probably binds elsewhere on the Csm1 C-terminal domain.
To confirm and extend these results, we purified the fourprotein MIND (Mtw1 including Nsl1, Nnf1, and Dsn1) kinetochore
complex (De Wulf et al., 2003), which forms an extended
25-nm-long structure containing one copy of each subunit
(Table S2; Figure S3). In the Ni2+-affinity pull-down assay, this
purified complex bound full-length Csm1 and the isolated
C-terminal globular domain, and mutating Csm1 residues Y156
and L161 disrupted the interaction (Figure S3). Using size exclusion chromatography, we observed that the Csm1/Lrs4 complex
comigrated with purified MIND complex, although some dissociation occurred during the course of the experiment (Figures 3B
and 3C). When the two complexes were present in a 1:4 ratio
(Csm1/Lrs4:MIND), essentially all of the Csm1/Lrs4 comigrated
with MIND. When the complexes were present at 1:2 or 1:1
ratios, some Csm1/Lrs4 did not comigrate with MIND. Thus,
each Csm1/Lrs4 complex can probably interact with up to four
MIND complexes, meaning that each conserved surface patch
in the complex can independently interact with a partner protein
(Figure 3C).
Because the conserved Csm1 surface patch is shared in all
fungal Csm1/Pcs1 orthologs (Figure S1), Dsn1 and/or Mif2
may represent conserved binding partners for these proteins.
To test this idea, we performed Ni2+-affinity pull-down assays
using S. pombe Pcs1 as bait, and the S. pombe Dsn1 and Mif2
orthologs, Mis13 and Cnp3, as prey. We found that the S. pombe
Pcs1 C-terminal domain interacts with Mis13, and with both fulllength Cnp3 and the minimal Pcs1-binding fragment identified
previously, amino acids 130–270 (Figure 3D) (Tanaka et al.,
2009). Binding to both Mis13 and Cnp3 was disrupted by
mutations to Pcs1 residues Y197 and I202 (Figure 3D), which
correspond to S. cerevisiae Csm1 residues Y156 and L161,
respectively (Figure S1). Thus, S. cerevisiae Csm1 and S. pombe
Pcs1 bind orthologous kinetochore subunits through the
conserved surface patch on their C-terminal globular domains.
Csm1 Point Mutations Disrupt Sister Chromatid
Co-orientation in Meiosis I
Deletion of any single monopolin subunit in S. cerevisiae results
in mis-segregation of chromosomes in meiosis I (Petronczki
et al., 2006; Rabitsch et al., 2003; Toth et al., 2000). In order to
observe sister chromatid MT attachment geometry in meiosis I,
we used an S. cerevisiae strain bearing an array of TET operator
sequences inserted at the chromosome V centromere and also
expressing a fusion of GFP and the Tet repressor protein, which
binds to the operator sites (referred to as CENV-GFP) (Lee and
Amon, 2003). We introduced CSM1 point-mutations into a strain
Figure 3. Csm1/Pcs1 Binding to the Kinetochore Subunits Dsn1 and Mif2/CENP-C
(A) In vitro-expressed and [35]S-labeled Dsn1 (upper panel), Mif2 (middle), and Mam1 (lower) were incubated with purified His6-tagged Csm1 constructs or point
mutants (as indicated), the resulting complexes incubated with Ni2+-affinity resin, and bound proteins analyzed by SDS-PAGE. Point mutations were designed to
disrupt the hydrophobic conserved patch identified in Figure 1D. See Figure S3A for a similar analysis using purified S. cerevisiae MIND complex.
(B) Superose 6 size exclusion chromatography of 0.2 mg purified MIND (red) and Csm1/Lrs4 (CL; blue) complexes. The migration of molecular weight standards
are shown at top. Locations of each band are marked at left of each gel; stars indicate proteolytic products of Dsn1 (MIND; upper panel) or contaminating Hsp70
(Csm1/Lrs4; lower panel).
(C) Size exclusion chromatography of MIND/CL mixtures. 1CL:1MIND contained equimolar amounts of the two complete complexes, and 1CL:4MIND contained
equimolar amounts of Csm1 (four protomers per CL complex) and the MIND complex. In each mixture, a portion of CL comigrates with MIND, saturating at
one MIND complex per Csm1 protomer (1CL:4MIND). For theoretical curves assuming no interaction, see Figure S3B. CL binding does not significantly alter
the elution profile of MIND, potentially because of the extremely extended shape of the MIND complex (see Figures S3C and S3D).
(D) In vitro-expressed and [35]S-labeled S. pombe Mis13 (Dsn1 ortholog; upper panel), Cnp3 (Mif2 ortholog; middle), and Cnp3 residues 130–270 (lower) were
incubated with purified His6-tagged S. pombe Pcs1 C-terminal domain (residues 85–222) or point mutants (as indicated), the resulting complexes incubated
with Ni2+-affinity resin, and bound proteins analyzed by SDS-PAGE. All point mutations were in the context of the isolated Pcs1 C-terminal domain, as the
full-length protein was poorly behaved in this assay. Y197 and I202 correspond to S. cerevisiae Csm1 residues Y156 and L161, respectively (Figure S1).
See also Figure S3 and Table S2.
heterozygous for CENV-GFP and with the endogenous promoter
of CDC20 replaced by that of CLB2, to arrest the cells in metaphase I (Lee and Amon, 2003). The heterozygous CENV-GFP
marker allows a simple readout of sister chromatid attachment
geometry: when the monopolin complex is functioning properly
to co-orient sister kinetochores, the GFP signals from the
two marked sister centromeres should overlap. If the monopolin complex is compromised, sister kinetochores become
bi-oriented, and spindle forces pull the marked sister centromeres far enough apart to form two resolved foci (Lee and
Amon, 2003; Monje-Casas et al., 2007; Toth et al., 2000). We
introduced the three Csm1 point mutations that disrupt Dsn1/
Mif2 binding in vitro: Y156/E, L161/D, and L161/K. Two
of these, L161/D and L161/K, result in 45% and 41% GFP
dot separation, respectively, matching the severity of a MAM1
deletion (40% separation, see Figure 4A). The third mutation,
Y156/E, does not cause elevated levels of sister chromatid
bi-orientation in vivo, suggesting that the Y156/E mutant
retains some affinity for its binding partners at the kinetochore
that is not detected by our pull-down assay.
We next used strains carrying CENV-GFP on both homologs
and the wild-type CDC20 promoter (no metaphase I arrest) to
examine chromosome segregation fidelity through a complete
meiosis, by counting GFP signals in the spores of tetrads. In
95% of wild-type tetrads, all four spores contained GFP foci,
indicating that each spore had received a single copy of
Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc. 561
eliminates kinetochore binding. Overall, however, our results
show that mutations to the conserved surface patch of Csm1
cause significant errors in meiotic chromosome segregation,
most likely by disrupting Csm1-kinetochore interactions in
metaphase I.
Figure 4. Effects of Csm1 Point Mutations on S. cerevisiae Meiotic
Chromosome Segregation
(A) Yeast strains with heterozygous CENV-GFP and homozygous monopolin
mutations (as noted) were arrested in metaphase I (pCLB2-CDC20) (Lee and
Amon, 2003), and scored for sister chromatid bi-orientation (gray) versus
co-orientation (white). Statistical significance values versus wild-type are
indicated as stars (3 stars, p < 0.001).
(B) Yeast strains with CSM1 mutations or deletion and homozygous
CENV-GFP were sporulated and examined for chromosome V segregation
to spores (2 stars, p < 0.01; 3 stars, p < 0.001) and for spore viability (data in
Table S3).
See also Table S3.
chromosome V (5% faulty segregation; Figure 4B and Table S3).
In contrast, a CSM1 deletion resulted in nearly 80% of tetrads
with GFP dots in only three, two, or one spore, in close agreement with previous studies (Rabitsch et al., 2003). The three
CSM1 point-mutations all showed statistically significant
increases in faulty segregation over wild-type, but varied in
severity, with the Y156/E mutation having the mildest effect
(16.5% faulty segregation) and L161/D the strongest (58%
faulty segregation; Figure 4B). We found a similar range in the
effects of the point mutations on spore viability: 88% of spores
were viable in the Y156/E mutant, 34% in L161/K, and only
12% in L161/D (wild-type = 93% viable, Dcsm1 = 4% viable;
Figure 4B and Table S3). This range of phenotypes indicates
that each point mutation affects Csm1-kinetochore interactions
to a different degree, and that none of the mutations completely
562 Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc.
Csm1 Point Mutations Affect rDNA Silencing
In addition to acting at kinetochores, Csm1 and Lrs4 localize to
the nucleolus during interphase, where they participate in
rDNA silencing and the suppression of USCE (Huang et al.,
2006; Huang and Moazed, 2003). Our structure of the Csm1/
Lrs4 complex and our studies of its interactions with kinetochore
components suggest that it may also crosslink proteins associated with rDNA, potentially through interactions in the conserved
surface patch of Csm1. We therefore studied the effects of Csm1
conserved-patch mutations in two assays, one measuring
reporter gene silencing and another measuring USCE. To
measure rDNA silencing, we compared the growth of yeast
strains with an mURA3 reporter gene inserted at either the leu2
locus (which is not silenced) or at two locations in the nontranscribed regions of the rDNA repeat (NTS1 or NTS2; Figure 5A),
on either complete media or media lacking uracil. This assay
has previously shown that the Sir2 histone deacetylase is
required for silencing throughout the rDNA, while a network of
proteins associated with the replication-fork block sequence
(RFB in Figure 5A), including Fob1 and Tof2 as well as Csm1/
Lrs4, is required for silencing specifically in the NTS1 region
(Huang et al., 2006; Huang and Moazed, 2003). We found that
mutations to the conserved surface patch of Csm1 had the
same effect as a CSM1 deletion: silencing was completely lost
at NTS1 and partially lost at NTS2 (Figure 5B).
We next tested the rate of recombination resulting in USCE,
which is strongly inhibited in wild-type cells through multiple
mechanisms: a Sir2-mediated mechanism presumably dependent on the assembly of silenced chromatin, and tethering of
rDNA repeats to the nuclear periphery mediated by Csm1/
Lrs4, the inner nuclear membrane proteins Heh1 and Nur1,
and possibly condensins (Huang et al., 2006; Huang and
Moazed, 2003; Johzuka and Horiuchi, 2009; Kaeberlein et al.,
1999; Mekhail et al., 2008). We measured the rate of loss of an
ADE2 reporter gene embedded within the rDNA array and found
a greater than three-fold increase in USCE when either CSM1 or
LRS4 was deleted (Figure 5C; Table S4), in agreement with
previous results (Huang et al., 2006). In contrast, we found that
none of the CSM1 point-mutations significantly increased
USCE (Figure 5C).
These results indicate that the Csm1/Lrs4 complex may have
multiple, biochemically distinct roles in rDNA regulation. The
behavior of the CSM1 point mutants resembles that of a deletion
of TOF2, another member of the RFB-bound protein network. A
TOF2 deletion results in loss of rDNA silencing at NTS1, but has
much more modest effects on USCE than deletions of CSM1,
LRS4, or SIR2 (Huang et al., 2006; Figure 5C). Because previous
biochemical purifications of rDNA-associated protein complexes (Huang et al., 2006) and two-hybrid screens (Wong
et al., 2007; Wysocka et al., 2004; Yu et al., 2008) had identified
Tof2 as a potential binding partner of Csm1, we tested this
interaction in vitro using Ni2+-affinity pull-downs. We found that
Figure 5. Csm1 Binds the rDNA Protein Tof2, and Csm1 Mutations Affect rDNA Silencing
(A) Diagram of a single rDNA repeat, with positions of mURA3 markers inserted into the array at NTS1 and NTS2 noted. 35S and 5S refer to ribosomal genes, RFB;
replication fork block sequence, rARS; autonomously replicating sequence.
(B) Silencing of an inserted mURA3 marker (at leu2, NTS1, or NTS2 as noted) was assessed by growth on synthetic complete media or media lacking uracil as
previously described (Huang et al., 2006).
(C) The rate of loss through unequal recombination of an ADE2 marker inserted into the rDNA array was measured as described previously (Huang et al., 2006;
Kaeberlein et al., 1999). For exact values, see Table S4.
(D) In vitro-expressed and [35]S-labeled Tof2 constructs were incubated with His-tagged wild-type and point mutant Csm1 proteins, and analyzed as in Figure 3.
(E) The localization of HA-tagged Lrs4 was examined in interphase, in wild-type and strains with CSM1 point-mutations or a TOF2 deletion. The characteristic
nucleolar localization of Lrs4 is lost in all of the mutant strains, although some residual nucleolar enrichment is visible in the TOF2 deletion strain.
See also Table S4.
full-length Tof2 specifically interacts with the Csm1 C-terminal
domain and that this binding is modestly affected by mutations
to the conserved surface patch (Figure 5D). Because full-length
Tof2 may be poorly behaved in solution, we also looked for truncations of Tof2 that bind Csm1. We identified a region (residues
251–500) that interacts strongly with wild-type Csm1 and
binding of which is disrupted by mutations to conserved-patch
residues Y156, L161, and K174 (Figure 5D). The effect of the
Csm1 K174/E mutation on Tof2 binding is much more
pronounced than its effect on binding of either Dsn1 or Mif2
(Figure 3A). This result, along with the finding that the Y156/E
mutation strongly affects Csm1/Lrs4 function at rDNA but has
more modest effects at kinetochores, suggests that although
Csm1 binds its multiple partners through a common surface,
the details of each interaction probably differ.
The finding that Csm1 binds Tof2 through its conserved
surface patch, together with the parallels between the behavior
of the TOF2 deletion and the CSM1 point mutations in genetic
Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc. 563
Figure 6. Model for Monopolin Complex Function in S. cerevisiae
(A) In S. cerevisiae mitosis, sister kinetochores become attached to MTs extending from opposite spindle poles. Sister chromatids (gray lines) are held together by
cohesin complexes (yellow rings), and the kinetochores are located at the tips of pericentric chromatin loops. Kinetochores are structurally subdivided into inner,
linker, and outer protein layers (labeled).
(B) In meiosis I, sister kinetochores co-orient due to monopolin complex (blue/orange) binding to Mif2 (localized to the inner layer) and Dsn1 (linker layer),
effectively fusing their outer kinetochores to form a composite MT-binding site.
(C) During interphase, Csm1/Lrs4 binds to rDNA repeats through an rDNA-bound protein complex that includes Fob1, Tof2, and the Sir2-containing RENT
complex. Crosslinking of multiple rDNA repeats by Csm1/Lrs4 may aid silencing by localized Sir2.
(D) Csm1/Lrs4 likely contributes to the suppression of USCE through interactions with the inner-nuclear membrane proteins Heh1 and Nur1, and condensin
complexes. Interactions between this protein network and that controlling rDNA silencing (shown in gray) are currently unknown.
assays, indicates that Csm1/Lrs4 and Tof2 probably function
together to aid rDNA silencing. This idea fits with the observation
that Csm1/Lrs4 depends on Tof2 for specific association with
the NTS1 region of rDNA repeats, as measured by chromatin
immunoprecipitation (Huang et al., 2006). Nonetheless, the
lack of effect on USCE in the CSM1 point mutant strains indicates that there is probably a Tof2-independent function for
Csm1/Lrs4 in suppressing rDNA recombination. To determine
whether Csm1/Lrs4 nucleolar localization is maintained upon
disruption of the Csm1-Tof2 interaction, we examined Lrs4
localization during interphase in strains with either a deletion of
TOF2 or with CSM1 point mutations (Y156/E or L161/D).
Wild-type cells showed a pattern of Lrs4 staining characteristic
of nucleolar localization (Figure 5E). In contrast, the TOF2 deletion and CSM1 point mutant strains showed mislocalization of
Lrs4, with the protein found dispersed throughout the nucleus
(Figure 5E). In the TOF2 deletion strain, but not the CSM1 point
mutant strains, some cells showed visible enrichment of Lrs4
in a nuclear region that stained poorly with DAPI, suggesting
that Csm1/Lrs4 may still be partially localized to the nucleolus
in the absence of interactions with Tof2. The more complete
dispersal of Lrs4 in the CSM1 point mutant strains as compared
to the TOF2 deletion also suggests that the Csm1 conserved
surface patch may mediate interactions with multiple partners
in the nucleolus, as it does at kinetochores.
DISCUSSION
The eukaryotic kinetochore is a large multiprotein structure
that creates a dynamic connection between chromosomes
564 Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc.
and MTs. The most likely candidate for direct MT binding in the
outer kinetochore is the 57-nm-long Ndc80 complex (Ciferri
et al., 2008; Wei et al., 2007), of which there are 6–8 copies in
each S. cerevisiae kinetochore (Joglekar et al., 2006). These
complexes cooperate to bind a single MT and extend outward
from a ‘‘linker’’ layer of protein complexes, including the
MIND/Mis12 complex, which in turn bind inner kinetochore
components, including the specialized Cse4/CENP-A histone
and the DNA-binding Mif2/CENP-C protein (Cheeseman et al.,
2006; Joglekar et al., 2006; Wei et al., 2007; Wei et al., 2005).
A single MT is 25 nm wide, and the V-shaped Csm1/Lrs4
complex presents two pairs of kinetochore-binding globular
domains separated by 10 nm. A plausible model for the mechanism of sister chromatid co-orientation by monopolin, then, is
that these two pairs of globular domains bind across sister kinetochores, bringing them so close together that they effectively
fuse and create a single, composite MT-binding site (Figures 6A
and 6B). This picture of kinetochore fusion is consistent with
measures of spindle MT numbers indicating that, in meiosis I,
each pair of sister kinetochores probably attaches to only a single
MT (Winey et al., 2005). We cannot currently speculate about the
exact geometry of sister kinetochore fusion: each Csm1/Lrs4
complex can probably bind four partners, and each of its kinetochore binding partners is present in multiple copies at each
kinetochore (two copies of Mif2, 6 copies of Dsn1) (Joglekar
et al., 2006). Thus, the interactions between Csm1/Lrs4 and
kinetochores are likely to be complex and stochastic, resulting
in promiscuous crosslinking of nearby elements both within individual kinetochores and between sister kinetochores. Moreover,
the relative importance of Csm1 interactions with its two thus-far
identified kinetochore binding partners, Dsn1 and Mif2, remains
uncertain. Finally, it is also unknown how Mam1, which binds the
Csm1 globular domain and is required for robust Csm1/Lrs4
localization to kinetochores (Rabitsch et al., 2003), might regulate or strengthen specific interactions between Csm1 and its
binding partners at the kinetochore.
S. pombe Pcs1 has overall sequence similarity to S. cerevisiae
Csm1, with high conservation in the kinetochore-binding surface
patch. We have shown that the Pcs1/Mde4 complex has the
same general architecture as S. cerevisiae Csm1/Lrs4 and that
it interacts with orthologous binding partners (Dsn1/Mis13 and
Mif2/Cnp3). From this evidence, we envision a crosslinking
mechanism for monopolin in S. pombe that is conceptually
similar to its mechanism in S. cerevisiae, but functioning within
a single kinetochore rather than across sister chromatids. The
kinetochores of S. pombe are larger than those of S. cerevisiae,
and they attach to 2-4 MTs through about 20 Ndc80 complexes
(Ding et al., 1993; Joglekar et al., 2008). In this context, monopolin-mediated crosslinking of inner kinetochore elements could
organize the outer kinetochore and force co-orientation of
adjacent MT-binding sites.
Our structural and functional data also help clarify the functions of Csm1/Lrs4 in maintaining the integrity of rDNA. Together
with previous data (Huang et al., 2006), our results indicate that
the Csm1/Lrs4 complex is recruited to the NTS1 region of
rDNA repeats through a direct interaction with Tof2, and that
this interaction is important for rDNA silencing. Because a deletion of Tof2 does not significantly affect association of Sir2 with
the rDNA (Huang et al., 2006), the loss of rDNA silencing in our
mutants cannot be due to simple loss of Sir2 localization. We
propose that Csm1/Lrs4 crosslinks multiple rDNA repeats
(up to four per Csm1/Lrs4 complex) through interactions with
rDNA-associated Tof2, thereby assisting localized Sir2 in
silencing these now closely juxtaposed/clustered repeats
(Figure 6C). This rDNA repeat clustering/crosslinking by Csm1/
Lrs4 and Tof2 must not, however, directly suppress unequal
sister chromatid exchange, because disrupting the Csm1-Tof2
interaction does not affect the rate of USCE. We propose that,
instead, Csm1/Lrs4 may control recombination through interactions with a different set of proteins: the inner nuclear membrane
proteins Heh1 and Nur1 (Mekhail et al., 2008) and condensin
complexes (Johzuka and Horiuchi, 2009). These interactions
mediate the tethering of rDNA repeats to the nuclear periphery
and may also link sister chromatids together in-register to suppress unequal exchange (Figure 6D). There is probably some
functional interplay between these distinct protein networks
controlling rDNA silencing and USCE. The structures and interactions we have described now provide a basis for determining
how Csm1/Lrs4 contributes to the activities of each of these
protein networks and to their interactions at rDNA.
EXPERIMENTAL PROCEDURES
were spun in a Beckman Optima XL-A centrifuge at three speeds, which varied
with the expected molecular weight of the protein/complex.
Crystallization and Structure Determination
Protein crystallization is described in the Extended Experimental Procedures.
All datasets were collected on NE-CAT beamlines 24ID-C and 24ID-E at the
Advanced Photon Source at Argonne National Laboratory. The structure of
Csm1 69–181 was determined by single-wavelength anomalous dispersion
(SAD) in space group R3 at 2.6 Å resolution, from a selenomethione-labeled
crystal of Csm1 69–181 (L157M), then determined by molecular replacement
in space group P21212 at 2.35 Å resolution. The structure of full-length
Csm1 was determined by molecular replacement in space group P3121 to
3.4 Å resolution. The Csm1 1–181/Lrs4 1–102(D38–44) complex structure
was determined by molecular replacement. Because of the low resolution
(6.0/3.9 Å) of the data, refinement was limited to rigid-body and restrained
B-factor refinement (see Table S1 for data and refinement statistics).
Electron Microscopy
Native Csm1/Lrs4 was purified as described (Huang et al., 2006), adsorbed to
glow-discharged carbon-coated copper grids, and stained with 0.75% (w/v)
uranyl formate (Ohi et al., 2004). Images were collected with a Tecnai T12
electron microscope (FEI, Hillsboro, OR) operated at 120 kV using low-dose
procedures, and processed using SPIDER software (Frank et al., 1996).
Ni2+ Affinity Pull-Down Assay
Bait proteins were purified as described above with His6-tags left intact, and
[35]
S-labeled prey proteins were produced using an in vitro coupled transcription/translation kit (Promega). Pull-down assays were performed essentially as
described (Rabitsch et al., 2003).
Yeast Strains, Sporulation, and Immunofluorescence
Strains were generated with PCR-based methods as described (Longtine
et al., 1998). For spore viability, cells were grown on YPD agar and then
patched onto SPO medium (1% KOAc) for 48–72 hr. Forty tetrads (160 spores)
were dissected for each strain. For metaphase I bi-orientation, cells were
grown in YPD, then diluted into BYTA (YEP plus 1% KOAc/50 mM potassium
phthalate) at an OD600 of 0.3, grown overnight, then washed and resuspended
in SPO medium (0.3% KOAc [pH 7.0]) at an OD600 of 2.0 at 30 C to induce
sporulation. Samples were removed hourly for 10 hr, fixed, and stained with
DAPI (bi-orientation was assayed at 7 hr); arrested cells were scored for one
(indicating co-orientation) or two (indicating bi-orientation) CENV-GFP foci
per nucleus. For CENV-GFP segregation to spores, cells were patched onto
SPO medium for 48–72 hr, then fixed and stained with DAPI. Tetrads were assayed for GFP foci in DAPI masses. Samples were compared to wild-type
using an independent two-sample t test. Indirect immunofluorescence and
chromosome spreads were performed as described previously (Visintin
et al., 1999).
rDNA Assays
rDNA silencing and unequal sister chromatid exchange assays were performed essentially as described (Huang et al., 2006; Huang and Moazed,
2003; Kaeberlein et al., 1999).
ACCESSION NUMBERS
Coordinates and structure factors for the reported crystal structures have
been deposited with the Protein Data Bank under accession codes 3N4R
(Csm1 69–181, R3 form), 3N4S (Csm1 69–181, P21212 form), 3N4X (Csm1
full-length), and 3N7N (Csm1/Lrs4 1–102).
For detailed methods, see the Extended Experimental Procedures.
SUPPLEMENTAL INFORMATION
Protein Expression and Purification
All proteins were expressed in Esherichia coli with TEV-protease cleavable
His6 tags, and purified by Ni2+, ion-exchange, and gel filtration chromatography. For sedimentation equilibrium analytical ultracentrifugation, proteins
Supplemental Information includes Extended Experimental Procedures,
three figures, and six tables and can be found with this article online at
doi:10.1016/j.cell.2010.07.017.
Cell 142, 556–567, August 20, 2010 ª2010 Elsevier Inc. 565
ACKNOWLEDGMENTS
We thank the staff at the Advanced Photon Source NE-CAT beamlines for
advice and assistance with data collection and interpretation; D. Moazed for
the gift of S. cerevisiae strains and valuable advice; M. Sawaya for providing
advice and script files for anisotropic scaling; J. Al-Bassam for assistance
with electron microscopy; members of the Amon laboratory, especially I. Brito
and E. Unal, for advice, assistance with yeast strain construction, and light
microscopy; and members of the Harrison laboratory and I. Cheeseman for
helpful discussions. K.D.C. acknowledges a postdoctoral fellowship from
the Helen Hay Whitney Foundation; C.K.Y. acknowledges a postdoctoral
fellowship from the Jane Coffin Childs Memorial Fund for Medical Research;
T.W., A.A, and S.C.H. are Investigators in the Howard Hughes Medical
Institute.
Received: December 1, 2009
Revised: May 14, 2010
Accepted: July 8, 2010
Published: August 19, 2010
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